Breast cancer is a highly heterogeneous disease with five major molecular subtypes distinguished by gene and protein expression. While treatments targeting endocrine functions of luminal A and luminal B breast cancers, and antibody therapy of Her2 positive tumors have reduced patient mortality in these groups, patients with triple negative breast cancers (TNBC) lacking estrogen and progesterone receptors as well as HER2, have no access to targeted therapies. The goal of this project is to identify novel targets in TNBC with potentially therapeutic and diagnostic antibodies. This will be accomplished by combining novel approaches to elucidate the antibody fingerprint of triple negative breast cancer cells through screening against spatially addressed human germline IgG and bovine antibody libraries. We generated a human germline antibody library with rationally designed structural diversity. To further enhance antibody diversity and enable unique discovery of otherwise hard-to-target antigens, we included cow antibody structures. While the majority of mammalian antibodies bind antigens with six CDR loops, arranged to form a flat binding surface, we recently identified ultra-long CDR H3 regions from cow antibodies that have a protruding stalk and knob architecture. This structure is highly diverse and allows specific recognition of antigen cavities, enzyme active sites, pores or channels, or unique conformational epitopes in complex targets. To elucidate the antibody fingerprint of TNBC cells, we defined two Specific Aims: Aim 1: Generate antibody fingerprints against breast cancer cell lines by high throughput flow cytometry and identify antigen-antibody pairs specific for TNBC cells. A panel of breast cancer cell lines from each of the 5 classes of breast cancer and normal breast epithelial cells will be screened against the human germline and cow antibody libraries. TNBC-specific antibodies will be identified, tested for binding specificity and affinity, and affinity matured if desired. We established feasibility and robustness of the approach for hard-to-target proteins such as GPCRs with multispanning transmembrane regions expressed on HEK293 cells. Aim 2: Use antibody fingerprints to identify targets and evaluate therapeutic and diagnostic potential of antibodies specific for triple negative breast cancer cells. Antibody targes will be determined by proteomic analyses. Reactivity and target specificity for TNBC will be assessed by antibody screening on clinical breast cancer samples. Functional properties of TNBC specific antibodies will be tested for endogenous cell growth inhibition. Antibody drug conjugates will be evaluated for direct target cell killing. Suitability of antibodies for T- cell directed therapy can be analyzed after generating bispecific antibody versions. Our study could identify clinically relevant targets on triple negative breast cancer cells, and simultaneously provide antibodies with therapeutic and diagnostic potential for future evaluation and development.